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Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondria! Protein Kgd4
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 52019 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, no 7, p. 1460-1467Article in journal (Refereed) Published
Abstract [en]

Kgd4 is a novel subunit of the mitochondria! a-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

Place, publisher, year, edition, pages
2019. Vol. 431, no 7, p. 1460-1467
Keywords [en]
mitochondria, translation, alternative initiation, protein import, biogenesis
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-169298DOI: 10.1016/j.jmb.2019.02.023ISI: 000464771800011PubMedID: 30822412OAI: oai:DiVA.org:su-169298DiVA, id: diva2:1320057
Available from: 2019-06-04 Created: 2019-06-04 Last updated: 2019-08-19Bibliographically approved
In thesis
1. Structure and Biogenesis of Membrane Proteins
Open this publication in new window or tab >>Structure and Biogenesis of Membrane Proteins
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins make up about one-third of the cellular proteome. The diverse roles that membrane proteins have in cells include major life-sustaining processes, making them major drug targets. The respiratory chain comprises a series of complexes of membrane proteins residing in the inner mitochondrial membrane, which serve as major drivers of ATP synthesis. Assembly of the respiratory chain complexes (RCC) requires coordinated synthesis of nuclear and mitochondrial subunits. Cbp3-Cbp6 complex binds to the mitoribosome as translational activator for cytochrome b synthesis and binds the nascent polypeptide to facilitate its hemylation. Cbp3 consists of an N-terminal domain specific to mitochondrial homologues and a conserved C-terminal ubiquinol-cytochrome c chaperone domain. In this thesis I present the first crystal structure of the C-terminal domain from a bacterial homologue that has enabled us to identify the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b using site-specific photo-crosslinking. Our finding suggests that Cbp3 contacts the mitoribosome via the N-terminal domain in a manner that positions the substrate binding site close to the tunnel exit. In the second project, we have analyzed the effects of disease causing cytochrome b mutations, on bc1 complex assembly. We found that complex III assembly is blocked at either intermediate 0 or I due to impaired insertion of bL or bH heme respectively, which indicates that assembly processes are involved in disease development. We then focused on NADH; a product of alpha-ketoglutarate dehydrogenase complex (KGDH) catalyzed citric acid cycle reaction and one of the substrates that supply electron to the respiratory chain. Kgd4 is a novel subunit of this enzyme complex and two functional variants (Kgd4S and Kgd4L) of unknown origins exist in yeast. We report in our work that Kgd4L originates from a UUG alternative start site, 90 nucleotides upstream and in frame of the annotated start codon. The sequence context upstream of UUG determines the efficiency of recognition of this alternative start codon. Finally, Na+/H+ antiporters are present in all species and are involved in regulation of intracellular pH, cell volume and sodium concentration. ATP formed during oxidative phosphorylation serves as energy source for Na+/K+ ATPase to generate Na+ gradient across the inner mitochondrial membrane, which drives local Na+/H+ antiporters. We show that K305 is involved in proton transport and responsible for the electrogenicity of NapA, while human NHA2 shows electroneutral antiporter activity.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 53
Keywords
Cbp3, cytochrome b, respiratory complex III, alternative translation initiation and sodium/proton exchange
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-171519 (URN)978-91-7797-749-0 (ISBN)978-91-7797-750-6 (ISBN)
Public defence
2019-09-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 2: Manuscript.

Available from: 2019-09-03 Created: 2019-08-13 Last updated: 2019-08-26Bibliographically approved

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